乳腺癌CD4^+ CD25^(high) Foxp^(3+)调节性T细胞数量和分布及Foxp3mRNA表达与肿瘤分期的相关性研究

The populations,distribution of CD4^+ CD25^(high) Foxp^(3+) regulatory T cells,FoxP3 mRNA expression breast cancer patients and the association with malignant stage

目的研究乳腺癌患者CD4+CD25high Foxp3+调节性T细胞(T regulatory cell,Treg)的存在数量、分布情况,及其功能基因Foxp3mRNA的表达变化与国际抗癌联盟(UICC)乳腺癌的TNM分期的关系。方法用四色流式细胞术以Foxp3-FITC/CD25-PE/CD4-PerCP/CD3-PC7抗体组合分析40名健康对照者及40例乳腺癌患者(临床TNM分期T1期9例、T2期10例、T3期12例、T4期9例)外周血、肿瘤组织浸润淋巴细胞(TIL)及癌旁淋巴结中CD4+CD25high Foxp3+Treg的数量及分布情况。实时荧光定量PCR技术检测TregFoxp3mRNA表达水平。结果乳腺癌患者外周血CD4+CD25high Foxp3+Treg占CD4T细胞的百分比为(8.91±3.06)%,高于正常健康对照组(5.84±2.63)%,差异具有统计学意义(P<0.05)。乳腺癌患者TIL、癌旁淋巴结中Treg占CD4+T淋巴细胞比例均高于外周血,差异有统计学意义(P均<0.01)。乳腺癌组织TIL和癌旁淋巴结中Treg的Foxp3平均荧光强度(MFI)显著高于外周血(P<0.05)。T1、T2、T3、T4期乳腺癌患者Treg比例分别为(5.81±1.89)%、(6.52±2.15)%、(9.65±3.14)%、(11.86±2.43)%。乳腺癌患者外周血单个核细胞Foxp3mRNA表达水平明显高于健康对照组(P<0.01)。相关分析显示,Treg数量与肿瘤TNM分期显著相关(r=0.7 576,P<0.01);Foxp3基因mRNA表达水平与乳腺癌TNM各分期呈显著相关性(r=0.6 129,P<0.05)。结论乳腺癌患者CD4+CD25high Foxp3+Treg的数量升高,其增高程度与肿瘤临床分期呈显著正相关。乳腺癌患者外周血和癌组织局部Treg的数量、分布情况及特异性转录因子Foxp3mRNA的表达强度均有所不同,提示这可能是产生肿瘤免疫抑制的重要机制,CD4+CD25high Foxp3+Treg将来可作为乳腺癌患者病情进展的重要判断指标之一。

Objective To investigate the populations of CD4＋CD25high Foxp3＋ regulatory T cells（Treg cells）and mRNA expression of Foxp3 in peripheral and the marginal region of tumor from patients with breast cancer,and to evaluate the prevalence of Treg cells from patients with breast cancer in relation to the TNM stages.Methods PBMC,tumor-infiltration lymphocyte and tumor-draining lymphnodes in 40 patients with breast cancer（TNM T19,T2 10,T312,T49）and PBMC in 40 normal healthy donors were evaluated for the proportion of Treg cells,as a percentage of the total CD4＋cells,by flow cytometric analysis.Levels of mRNA for Foxp3 were measured with a real-time quantitative PCR.Results The percentage of CD4＋CD25high Foxp3＋ Treg cells in PBMC for cases of breast cancer were significantly higher than those for healthy donors（P0.05）.The percentage of Treg cells were more abundant in TIL and tumor draining lymphnodes（Pall0.01）of individuals with breast cancer than in their blood.And the Foxp3 mean fluorescent intensity（MFI）of Treg cells in TIL and tumor draining lymphnodes with breast cancer were significantly higher than PBMC P0.05）.The proportion of Treg cell in patients of breast cancer with stage T1、T2、T3、T4 were（5.81±1.89）%、（6.52±2.15）%、（9.65±3.14）%、（11.86±2.43）%,respectively.There were significant correlation between prevalence of Treg cells and disease stages in breast cancer（r=0.7 576,P0.01）.Foxp3 mRNA levels were higher in PBMC from the group with breast cancers than in the group with healthy donors.Foxp3 mRNA levels were correlated with TNM stages（r=0.6 129,P0.05）.Conclusion Our results suggest that the populations of CD4＋CD25high Foxp3＋ Treg cells and Foxp3 mRNA levels in patients with breast cancer are significantly higher in comparison to those in control donors.In addition,the expression of Foxp3 mRNA increased with tumor stage.The increased prevalence of T reg cells may be one of the explanations for impaired cell-mediated immunity in cancer bearing hosts.